Abstract We have discovered a family of novel secreted kinases that are key mediators of a variety of cellular processes including differentiation, mineralization, and extracellular signaling. These kinases share little sequence identity with canonical kinases and function extracellularly and/or in the lumen of the secretory pathway. We have determined that Fam20C is the authentic Golgi casein kinase that phosphorylates casein and other secreted proteins involved in mineralization within Ser-x-Glu (S-x-E) motifs. Phosphoproteomic studies have established that greater than 75% of secreted phosphoproteins are phosphorylated on S-x-E motifs, thus making Fam20C an important potential regulator of numerous physiological functions. This proposal is focused on understanding the substrate specificity, evolution, and structure/function of Fam20C. Specific Aim 1 is an in depth biochemical characterization of Fam20C, including a detailed kinetic analysis and identification of novel substrates. Specific Aim 2 addresses the evolution and functional conservation of Fam20 kinases in Caenorhabditis elegans and Drosophila melanogaster, two organisms lacking mineralized tissue. Using bioinformatics we have identified potential substrates for C. elegans and Drosophila Fam20 enzymes that will shed new light on the evolutionary origins of secreted protein phosphorylation. The goal of Specific Aim 3 is to solve the crystal structure of Fam20C. We have made significant progress toward obtaining the structure of the C. elegans Fam20C ortholog and have obtained crystals that diffract to 2.6. We propose to solve structures for C. elegans Fam20 complexed with its co-substrates. In addition, we will pursue the structure of Fam20B, a closely related family member that does not phosphorylate S-x-E, but utilizes a distinct proteoglycan substrate. Successful completion of these specific aims will result in a better understanding of this important family of novel secreted kinases.